The invention relates to the Ikaros gene and to the differentiation and generation of T cells.
The generation of the T cell repertoire from a progenitor stem cell proceeds through a differentiation pathway in which the later intrathymic steps are well documented while the early extrathymic events are only poorly characterized. One of the earliest definitive T cell differentiation markers is the CD3xcex4 gene of the CD3/TCR complex.
The Ikaros gene, a gene active in the early differentiation of lymphocytes, e.g. T cells and B cells, has been discovered. The gene encodes a family of unique zinc finger proteins, the Ikaros proteins. The proteins of the Ikaros family are isoforms which arise from differential splicing of Ikaros gene transcripts. The isoforms of the Ikaros family generally include a common 3xe2x80x2 exon (Ikaros exon E7, which includes amino acid residues 283-518 of the mouse Ikaros protein represented by SEQ ID No. 5, and amino acid residues 229-461 of the human Ikaros protein represented by SEQ ID No. 3) but differ in the 5xe2x80x2 region. The Ikaros family includes all naturally occurring splicing variants which arise from transcription and processing of the Ikaros gene. Eight such isoforms are described herein. The Ikaros family may also includes other isoforms, including those generated by mutagenesis and/or by in vitro exon shuffling. The naturally occurring Ikaros proteins can bind and activate (to differing extents) the enhancer of the CD3xcex4 gene, and are expressed primarily if not solely in T cells in the adult. The expression pattern of this transcription factor during embryonic development suggests that Ikaros proteins play a role as a genetic switch regulating entry into the T cell lineage. The Ikaros gene is also expressed in the proximal corpus striatum during early embryogenesis in mice.
In general, the invention features, nucleic acid, e.g., DNA, preferably a purified DNA, including (or consisting essentially of) a sequence which encodes a peptide including (or consisting essentially of) one or more Ikaros exons. In preferred embodiments: the Ikaros exon is any of E1/2, E3, E4, E5, E6, or E7; the purified DNA does not encode exon E7.
In other preferred embodiments: the encoded peptide further includes a second Ikaros exon; the second exon is any of E1/2, E3, E4, E5, E6, or E7; the first exon is E7 and the second exon is any of E1/2, E3, E4, E5, E6.
In other preferred embodiments: the encoded peptide further includes a third Ikaros exon; the third exon is any of E1/2, E3, E4, E5, E6, or E7; the first exon is E7, said second exon is E3, and the third exon is E1/2; the peptide is Ikaros isoform 5.
In other preferred embodiments: the encoded peptide further includes a fourth Ikaros exon; the fourth exon is any of E1/2, E3, E4, E5, E6, or E7; the first exon is E7, the second exon is E6, the third exon is E4, and the fourth exon is E1/2; the first exon is E7 , the second exon is E4, the third exon is E3, and the fourth exon is E1/2; the peptide is Ikaros isoform 3 or 4.
In other preferred embodiments: the encoded peptide further includes a fifth Ikaros exon; the fifth exon is any of E 1/2, E3, E4, E5, E6, or E7; the first exon is E7, the second exon is E6, the third exon is E5, the fourth exon is E4, and the fifth exon is E1/2; the peptide is Ikaros isoform 2.
In preferred embodiments: the encoded peptide further includes a sixth Ikaros exon; the sixth exon is any of E1/2, E3, E4, E5, E6, or E7; the first exon is E7, the second exon is E6, the third exon is E5, the fourth exon is E4, the fifth exon is E3, and the sixth exon is E1/2; the peptide is Ikaros isoform 1.
In preferred embodiments: the sequence of the encoded Ikaros exon is essentially the same as that of a naturally occurring Ikaros exon, or a fragment thereof having Ikaros activity; the DNA sequence which encodes the Ikaros exon is at least 85%, more preferably at least 90%, yet more preferably at least 95%, and most preferably at least 98 or 99% homologous with DNA encoding a naturally occurring Ikaros exon, or a fragment thereof having Ikaros activity, e.g., Ikaros exon encoded by DNA from any of SEQ ID NOS: 2-8 or SEQ ID NO:165; the sequence which encodes an Ikaros exon hybridizes under high or low stringency to a nucleic acid which encodes a naturally occurring Ikaros exon, or a fragment thereof having Ikaros activity, e.g., an Ikaros exon with the same, or essentially the same, amino acid sequence as an Ikaros exon of any of SEQ ID NOS:2-8 or SEQ ID NO: 153 or SEQ ID NO:165; the amino acid sequence of the encoded Ikaros exon is at least 30, more preferably at least 40, more preferably at least 50, and most preferably at least 60, 80, 100, or 200 amino acid residues in length; the encoded Ikaros amino acid sequence is at least 50% more preferably 60%, more preferably 70%, more preferably 80%, more preferably 90%, and most preferably 95% as long as a naturally occurring Ikaros exon, or a fragment thereof having Ikaros activity; the encoded Ikaros exon is essentially equal in length to a naturally occurring Ikaros exon, or a fragment thereof having Ikaros activity; the amino acid sequence of the encoded Ikaros exon is at least 80%, more preferably at least 85%, yet more preferably at least 90%, yet more preferably at least 95%, and a most preferably at least 98 or 99% homologous with a naturally occurring Ikaros exon sequence, or a fragment thereof having Ikaros activity, e.g., an Ikaros exon sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8 or SEQ ID NO:165; the encoded Ikaros exon amino acid sequence is the same, or essentially the same, as that of a naturally occurring Ikaros exon, or a fragment of the sequence thereof, e.g., an Ikaros exon described in any of SEQ ID NOS:2-8 or SEQ ID NO:165, and the peptide has Ikaros peptide activity.
In preferred embodiments the Ikaros encoding DNA includes at least two exons and: the DNA can be represented by the general formula A-B-C-D-E, wherein A represents Exon 3 or is absent, B represents Exon 4 or is absent, C represents Exon 5 or is absent, D represents Exon 6 or is absent, and E represents Exon 7 or is absent; the polypeptide includes at least two of said exons; the encoded polypeptide includes at least one exon containing a zinc finger domain; the encoded polypeptide includes at least one exon selected from E3, E4 or E5.
In other embodiments, the Ikaros encoding DNA includes a sequence represented by the general formula {Ex1-Ex2 . . . Exn} wherein each of Ex1 through Exn represents any of the Ikaros Exons 1/2, 3, 4, 5, 6 or 7, and n is an integer from zero to 10, more preferably an integer from zero to 5, In preferred embodiments: the polypeptide is a combination of 2 or more Ikaros exons, the combination of which may or may not naturally occur; the polypeptide includes at least two of said exons and is represented by the formula Ex1-Ex2; the polypeptide includes at least three of said exons and is represented by the formula Ex1-Ex2-Ex3; the polypeptide includes at least four of said exons and is represented by the formula Ex1-Ex2-Ex3-Ex4; the polypeptide includes at least five of said exons and is represented by the formula Ex1-Ex2-Ex3-Ex4-Ex5; the polypeptide includes at least six of said exons and is represented by the formula Ex1-Ex2-Ex3-Ex4-Ex5-Ex6; the polypeptide includes at least one exon containing a zinc finger domain; the polypeptide includes at least one exon selected from E3, E4 or E5.
In preferred embodiments: the exons in the encoded peptide are arranged in the same relative linear order as found in a naturally occurring isoform, e.g., Ikaros isoform 1, e.g., in a peptide having the exons E3 and E7, E3 is located N-terminal to E7; the linear order of the encoded exons is different from that found in a naturally occurring isoform, e.g., in Ikaros isoform 1, e.g., in a peptide having exons E3, E5, and E7, the direction N-terminal to C-terminal end, is E5, E3, E7; the exons in the encoded peptide differ in one or more of composition (i.e., which exons are present), linear order, or number (i.e., how many exons are present or how many times a given exon is present) from a naturally occurring Ikaros isoform, e.g., from Ikaros isoform 1, 2, 3, 4, 5, 6, 7 or 8; e.g. the Ikaros isoform is generated by in vitro exon shuffling.
In preferred embodiments: the Ikaros polypeptide includes less than 4, 3, 2, or 1 functional N terminal Zinc finger domains (the N terminal Zinc finger domains include domains F1-4, the C terminal Zinc finger domains include domains F1-4); the Ikaros polypeptide includes one or two functional C terminal Zinc finger domains; the Ikaros polypeptide includes less than 4, 3, 2, or 1 functional N terminal Zinc finger domains and includes one or two functional C terminal Zinc finger domains.
In another preferred embodiment: the Ikaros polypeptide includes a point mutation in or a deletion for all or part of the DNA binding region, e.g., a point mutation in, or a deletion for all or part of one or more of F1, F2, F3, or F4; the Ikaros polypeptide includes a mutation in the DNA binding region, e.g., a point mutation in, or a deletion for all or part of one or more of F1, F2, F3, or F4.
Also included in the invention is a composition which includes an Ikaros polypeptide (or a nucleic acid which encodes it) and one or more additional components, e.g., a carrier, diluent, or solvent. The additional component can be one which renders the composition useful for in vitro, in vivo, pharmaceutical, or veterinary use.
In another aspect, the invention features, a peptide, preferably a substantially pure peptide, including (or consisting essentially of) one or more Ikaros exons. In preferred embodiments: the Ikaros exon is E1/2, E3, E4, E5, E6, or E7; the peptide does not include exon E7.
In other preferred embodiments: the peptide further includes a second Ikaros exon; the second exon is any of E1/2, E3, E4, E5, E6, or E7; the first exon is E7 and the second exon is any of E1/2, E3, E4, E5, E6.
In other preferred embodiments: the peptide further includes a third Ikaros exon; the third exon is any of E1/2, E3, E4, E5, E6, or E7; the first exon is E7, the second exon is E3, and the third exon is E1/2; the peptide is Ikaros isoform 5.
In other preferred embodiments: the peptide further includes a fourth Ikaros exon; the fourth exon is any of E1/2, E3, E4, E5, E6, or E7; the first exon is E7, the second exon is E4, the third exon is E3, and the fourth exon is E1/2; the first exon is E7, the second exon is E4, the third exon is E3, and the fourth exon is E1/2; the peptide is Ikaros isoform 3 or 4.
In other preferred embodiments: the peptide further includes a fifth Ikaros exon; the fifth exon is any of E1/2, E3, E4, E5, E6, or E7; the first exon is E7, the second exon is E6, the third exon is E5, the fourth exon is E4, and the fifth exon is El/2; the peptide is Ikaros Isoform 2.
In other preferred embodiments: the peptide further includes a sixth Ikaros exon; the sixth exon is any of E1/2, E3, E4, E5, E6, or E7; the first exon is E7, the second exon is E6, the third exon is E5, the fourth exon is E4, the fifth exon is E3, and the sixth exon is E1/2; the peptide is Ikaros isoform 1.
In preferred embodiments: the sequence of the Ikaros exon is essentially the same as that of a naturally occurring Ikaros exon, or a fragment thereof having Ikaros activity; the amino acid sequence of the Ikaros exon is such that a nucleic acid sequence which encodes it is at least 85%, more preferably at least 90%, yet more preferably at least 95%, and most preferably at least 98 or 99% homologous with a naturally occurring Ikaros exon, or a fragment thereof having Ikaros activity, e.g., Ikaros having an amino acid sequence represented in any of SEQ ID NOS:2-8 or SEQ ID NO:153 or SEQ ID NO:165; the amino acid sequence of the Ikaros exon is such that a nucleic acid sequence which encodes it hybridizes under high or low stringency to a nucleic acid which encodes a naturally occurring Ikaros exon, or a fragment thereof having Ikaros activity, e.g., an Ikaros exon with the same, or essentially the same, amino acid sequence as an Ikaros exon represented in any of SEQ ID NOS:2-8 or SEQ ID NO:165; the amino acid sequence of the Ikaros exon is at least 30, more preferably at least 40, more preferably at least 50, and most preferably at least 60, 80, 100, or 200 amino acid residues in length; the encoded Ikaros amino acid sequence is at least 50% more preferably 60%, more preferably 70%, more preferably 80%, more preferably 90%, and most preferably 95% as long as a naturally occurring Ikaros exon, or a fragment thereof having Ikaros activity; the Ikaros exon is essentially equal in length to a naturally occurring Ikaros exon; the amino acid sequence of the Ikaros exon is at least 80%, more preferably at least 85%, yet more preferably at least 90%, yet more preferably at least 95%, and a most preferably at least 98 or 99% homologous with a naturally occurring Ikaros exon sequence, or a fragment thereof having Ikaros activity, e.g., an Ikaros exon sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:165; the Ikaros exon amino acid sequence is the same, or essentially the same, as that of a naturally occurring Ikaros exon, or a fragment of the sequence thereof, e.g., an Ikaros exon described in any of SEQ ID NOS:2-8 or SEQ ID NO:165; and the peptide has Ikaros peptide activity; the peptide has Ikaros antagonist activity.
In preferred embodiments: the Ikaros protein comprises a polypeptide represented by the general formula A-B-C-D-E, wherein A represents Exon 3 or is absent, B represents Exon 4 or is absent, C represents Exon 5 or is absent, D represents Exon 6 or is absent, and E represents Exon 7 or is absent; the polypeptide includes at least two of said exons; the polypeptide includes at least one exon containing a zinc finger domain; the polypeptide includes at least one exon selected from E3, E4 or E5.
In preferred embodiments: the exons in the peptide are arranged in the same relative linear order as found in a naturally occurring isoform, e.g., in Ikaros isoform 1, e.g., in a peptide having the exons E3 and E7, E3 is located N-terminal to E7; the linear order of the exons is different from that found in a naturally occurring isoform, e.g., in Ikaros isoform 1, e.g., in a peptide having exons E3, E5, and E7, the direction N-terminal to C-terminal end, is E5, E3, E7; the exons in the peptide differ in one or more of composition (i.e., which exons are present), linear order, or number (i.e., how many exons are present or how many times a given exon is present) from a naturally occurring Ikaros isoform, e.g., from Ikaros isoform 1, 2, 3, 4, or 5; e.g. the Ikaros protein is an isoform generated by in vitro exon shuffling.
Another aspect the invention features a DNA, preferably a purified DNA, which includes (or consists essentially of) a DNA sequence encoding an Ikaros peptide, e.g., an Ikaros peptide having Ikaros activity, e.g., Ikaros isoform 1, 2, 3, 4, or 5, or an Ikaros peptide which is an antagonist of an Ikaros activity. In preferred embodiments: the sequence of the encoded Ikaros peptide is essentially the same as the sequence of a naturally occurring Ikaros peptide, or a fragment thereof having Ikaros activity; the DNA sequence is at least 85%, more preferably at least 90%, yet more preferably at least 95%, and most preferably at least 98 or 99% homologous with DNA encoding a naturally occurring Ikaros peptide, or a fragment thereof having Ikaros activity; e.g., with DNA from any of SEQ ID NOS:2-8 or SEQ ID NO:165; the amino acid sequence of the encoded peptide is such that it can be encoded by a nucleic acid which hybridizes under high or low stringency conditions to a nucleic acid which encodes a peptide with the same, or essentially the same, amino acid sequence as the peptide of any of SEQ ID NOS:2-8 or SEQ ID NO:153 or SEQ ID NO:165; the encoded peptide is at least 30, more preferably at least 40, more preferably at least 50, and most preferably at least 60, 80, 100, or 200 amino acid residues in length; the encoded peptide is at least 50% more preferably at least 60%, more preferably 70%, more preferably 80%, more preferably 90%, and most preferably 95% as long as a naturally occurring Ikaros peptide, or a fragment thereof having Ikaros activity; the encoded peptide is essentially the same length as a naturally occurring Ikaros peptide, or a fragment thereof having Ikaros activity; the encoded peptide is at least 80%, more preferably at least 85%, yet more preferably at least 90%, yet more preferably at least 95%, and a most preferably at least 98 or 99% homologous with an amino acid sequence which is the same, or essentially the same, as a naturally occurring Ikaros peptide, or a fragment thereof having Ikaros activity, e.g., the peptide sequence of any of SEQ ID NOS:2-8 or SEQ ID NO:153 or SEQ ID NO:165; and, the amino acid sequence of the peptide is essentially the same as the sequence of a naturally occurring Ikaros peptide, or a fragment thereof having Ikaros activity, e.g., the sequence, described in any of SEQ ID NOS:2-8 or SEQ ID NO: 153 or SEQ ID NO:165.
Another aspect, the invention features a DNA, preferably a purified DNA, which includes (or consists essentially of) a sequence encoding a peptide of 20 or more amino acids in length, the peptide having at least 90% homology with an amino acid sequence which is the same as the amino acid sequence of any of SEQ ID NOS:2-8, or SEQ ID NO:153 or SEQ ID NO:165. In preferred embodiments: the purified DNA encodes a peptide which is at least 30, more preferably at least 40, more preferably at least 50, and most preferably at least 60, 80, 100, or 200, amino acid residues in length; the encoded peptide is at least 50% more preferably at least 60%, more preferably 70%, more preferably 80%, more preferably 90%, and most preferably 95% as long as a naturally occurring Ikaros peptide, or fragment thereof having Ikaros activity; the encoded peptide is essentially the same length as a naturally occurring Ikaros peptide; a peptide which is at least 80, more preferably at least 85, yet more preferably at least 90, yet more preferably at least 95, and most preferably at least 98 or 99% homologous with an amino acid sequence which is the same, or essentially the same, as a naturally occurring Ikaros peptide, or a fragment thereof having Ikaros activity, e.g., as the amino acid sequence of any of SEQ ID NOS:2-8, SEQ ID NO:153 or SEQ ID NO:165; and, a peptide having one of either an Ikaros activity or an Ikaros antagonist activity.
In another aspect, the invention features, a DNA, preferably a purified DNA, which includes (or consists essentially of) a DNA sequence which hybridizes under high or low stringency to a nucleic acid which encodes a peptide with the same, or essentially the same, amino acid sequence as a naturally occurring Ikaros peptide, e.g., the peptide of any of SEQ ID NOS:2-8, or SEQ ID NO:153 or SEQ ID NO:165. In preferred embodiments: the DNA sequence is at least 85%, more preferably at least 90%, yet more preferably at least 95%, and most preferably at least 98 or 99% homologous with DNA encoding a naturally occurring Ikaros peptide, or a fragment thereof having Ikaros activity, e.g., with DNA from of any of SEQ ID NOS:2-8 or SEQ ID NO:165; the purified DNA encodes a peptide at least 30, more preferably at least 40, more preferably at least 50, and most preferably at least 60, 80, 100, or 200 amino acid residues in length; the encoded peptide is at least 50% more preferably at least 60%, more preferably 70%, more preferably 80%, more preferably 90%, and most preferably 95% as long as a naturally occurring Ikaros peptide, or fragment thereof having Ikaros activity; the encoded peptide is essentially the same length as a naturally occurring Ikaros peptide; the purified DNA encodes a peptide at least 80, more preferably at least 85, yet more preferably at least 90, yet more preferably at least 95, and most preferably at least 98 or 99% homologous with an amino acid sequence which is the same, or essentially the same, as a naturally occurring Ikaros peptide, e.g., the amino acid sequence of any of SEQ ID NOS:2-8 or SEQ ID NO:153 or SEQ ID NO:165; and, the purified DNA encodes a peptide having essentially the same amino acid sequence, or a fragment of the amino acid sequence, described in SEQ ID NOS:2-8 or SEQ ID NO:153 or SEQ ID NO:165.
In preferred embodiments: the encoded Ikaros polypeptide includes less than 4, 3, 2, or 1 functional N terminal Zinc finger domains (the N terminal Zinc finger domains include domains F1-4, the C terminal Zinc finger domains include domains F1-4); the encoded Ikaros polypeptide includes one or two functional C terminal Zinc finger domains; the encoded Ikaros polypeptide includes less than 4, 3, 2, or 1 functional N terminal Zinc finger domains and includes one or two functional C terminal Zinc finger domains.
In another preferred embodiment: the encoded Ikaros polypeptide includes a point mutation in or a deletion for all or part of the DNA binding region, e.g., a point mutation in, or a deletion for all or part of one or more of F1, F2, F3, or F4; the encoded Ikaros polypeptide includes a mutation in the DNA binding region, e.g., a point mutation in, or a deletion for all or part of one or more of F1, F2, F3, or F4.
In another aspect, the invention includes a vector which includes DNA of the invention, preferably a purified-DNA of the invention, which encodes a peptide of the invention.
The invention also includes: a cell, e.g., a cultured cell or a stem cell, containing purified Ikaros-protein-encoding-DNA; a cell capable of expressing an Ikaros protein; a cell capable of giving rise to a transgenic animal or to a homogeneous population of hemopoietic cells, e.g., lymphoid cells, e.g., T cells; an essentially homogeneous population of cells, each of which includes purified Ikaros-protein-encoding-DNA; and a method for manufacture of a peptide of the invention including culturing a cell which includes a DNA, preferably a purified DNA, of the invention in a medium to express the peptide.
In another aspect, the invention features a peptide of the invention, preferably a substantially pure peptide of the invention, e.g.: a peptide having Ikaros activity, e.g., Ikaros isoform 1, 2, 3, 4, 5, 6, 7 or 8; a peptide having Ikaros antagonistic activity, e.g. able to inhibit at least one biological activity of a naturally occurring Ikaros, e.g. any of isoforms or 1,2,3,4, 5, 6, 7 or 8. In preferred embodiments: the sequence of the encoded Ikaros peptide is essentially the same as the sequence of a naturally occurring Ikaros peptide, or a fragment thereof having Ikaros activity; the sequence of the peptide is such that it is encoded by a DNA sequence at least 85%, more preferably at least 90%, yet more preferably at least 95%, and most preferably at least 98 or 99% homologous with DNA encoding a naturally occurring Ikaros peptide, or a fragment thereof having Ikaros activity; e.g., with DNA from any of SEQ ID NOS:2-8 or SEQ ID NO:165; the amino acid sequence of the peptide having Ikaros activity or Ikaros antagonistic activity is such that it can be encoded by a nucleic acid which hybridizes under high or low stringency conditions to a nucleic acid which encodes a peptide with the same, or essentially the same, amino acid sequence as the peptide of any of SEQ ID NOS:2-8, or SEQ ID NO:153 or SEQ ID NO:165; the peptide is at least 30, more preferably at least 40, more preferably at least 50, and most preferably at least 60, 80, 100, or 200 amino acid residues in length; the peptide is at least 50% more preferably at least 60%, more preferably 70%. more preferably 80%, more preferably 90%, and most preferably 95% as long as a naturally occurring Ikaros peptide, or fragment thereof having Ikaros activity; the peptide is essentially the same length as a naturally occurring Ikaros peptide, or a fragment thereof having Ikaros activity; the peptide is at least 80%, more preferably at least 85%, yet more preferably at least 90%, yet more preferably at least 95%, and a most preferably at least 98 or 99% homologous with an amino acid sequence which is the same. or essentially the same, as a naturally occurring Ikaros peptide, or a fragment thereof having Ikaros activity, e.g., the peptide sequence of any of SEQ ID NOS:2-8, or SEQ ID NO:153 or SEQ ID NO:165; and, the amino acid sequence of the peptide is essentially the same as the sequence of a naturally occurring Ikaros peptide, or a fragment thereof having Ikaros activity, e.g., the sequence, described in SEQ ID NOS:2-8 or SEQ ID NO:165.
In preferred embodiments a peptide of the invention, preferably a purified peptide of the invention, is produced by expression of a DNA of the invention, preferably a purified DNA of the invention.
In another aspect, the present invention features recombinant Ikaros proteins which are encoded by genes derived from vertebrate organisms (e.g. a mammal, e.g. a human, a mouse or a pig) and which is capable of functioning in one of either role of an agonist or an antagonist of at least one biological activity of a naturally occurring Ikaros protein. The term xe2x80x9crecombinant proteinxe2x80x9d refers to a Ikaros protein of the present invention which is produced by recombinant DNA techniques, wherein generally DNA encoding the Ikaros protein is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein. Moreover, the phrase xe2x80x9cderived fromxe2x80x9d, with respect to a recombinant gene encoding the recombinant Ikaros, is meant to include within the meaning of xe2x80x9crecombinant proteinxe2x80x9d those proteins having an amino acid sequence of a naturally occurring Ikaros isoform, or an amino acid sequence similar thereto which is generated by, for example, mutations including substitutions and deletions of a naturally occurring Ikaros isoform. Recombinant proteins preferred by the present invention, in addition to native vertebrate Ikaros proteins, are at least 85%, more preferably at least 90%, yet more preferably at least 95%, and most preferably at least 98 or 99% homologous with an amino acid sequence selected from the group consisting of any of SEQ ID NOS: 2-8 or SEQ ID NO:165, e.g. a protein represented by the general formula of SEQ ID NO:153.
In particular, recombinant Ikaros protein, as used herein, includes a protein of the same or similar sequence as a naturally occurring Ikaros protein (e.g. a protein having an amino acid sequence found in any of SEQ ID NOS: 2-8 or SEQ ID NO:165) but lacking amino acid sequences at either or both of its N-terminal and C-terminal ends. Examples of such proteins include, but are not limited to, Ikaros isoforms which lack either exon 1, or exon 7, or both. In other exemplary embodiments, the recombinant proteins are truncation mutants. In preferred embodiments, the truncation mutants comprise at least 50-60 amino acid residues, more preferably 90-100 amino acid residues, and most preferably at least 150 amino acid residues of an Ikaros protein, or variant thereof, while retaining the activity of either an Ikaros agonist or an Ikaros antagonist.
The present invention further pertains to recombinant Ikaros proteins which are encoded by genes derived from a vertebrate organism and which have amino acid sequences evolutionarily related to naturally occurring Ikaros protein. Such recombinant Ikaros proteins preferably are capable of functioning in one of either role of an agonist of antagonist of at least one biological activity of a vertebrate Ikaros. The term xe2x80x9cevolutionarily related toxe2x80x9d, with respect to amino acid sequences of the present recombinant Ikaros proteins, refers to vertebrate Ikaros proteins having amino acid sequences which have arisen naturally. The term xe2x80x9cevolutionarily related toxe2x80x9d also refers to mutational variants of naturally occurring Ikaros proteins which are derived, for example, by combinatorial mutagenesis or in vitro exon shuffling. In an illustrative embodiment the recombinant Ikaros protein is an isoform encoded by a recombinant Ikaros gene generated through permutation of an exon order relative to a naturally occurring Ikaros protein, e.g. of SEQ ID NOS:2-8 or SEQ ID NO:165, e.g. of any exons 1/2, 3, 4, 5, 6, 7, e.g. wherein two different exons are permuted in sequential order relative to a naturally occurring Ikaros isoforms, e.g. when an Ikaros exon is present two or more times in the recombinant Ikaros gene.
The invention also includes substantially pure preparation of an antibody, preferably a monoclonal antibody directed against an Ikaros protein; a therapeutic composition including an Ikaros protein and a pharmaceutically acceptable carrier; a therapeutic composition which includes a purified DNA of the invention and a pharmaceutically acceptable carrier.
In another aspect, the invention features a method for treating an animal, e.g., a human, a mouse, a transgenic animal, or an animal model for an immune system disorder, e.g., a T or B cell related disorder, e.g., a nude mouse or a SCID mouse, including administering a therapeutically-effective amount of an Ikaros peptide to the animal.
In another aspect, the invention features a method for treating an animal, e.g., a human, a mouse, a transgenic animal, or an animal model for an immune system disorder, e.g., a T or B cell related disorder, e.g., a nude mouse or a SCID mouse including administering to the animal cells selected, e.g., selected in vitro, for the expression of a product of the Ikaros gene, e.g., hematopoietic stem cells, e.g., cells transformed with Ikaros-peptide-encoding DNA, e.g., hematopoietic stem cells transformed with Ikaros-peptide-encoding DNA.
In preferred embodiments: the cells are taken from the animal to which they are administered; the cells are taken from an animal which is MHC matched with the animal to which they are administered; the cells are taken from an animal which is syngeneic with the animal to which they are administered; the cells are taken from an animal which is of the same species as is the animal to which they are administered.
In another aspect, the invention features a method for treating an animal, e.g., a human, a mouse, a transgenic animal, or an animal model for an immune system disorder, e.g., a T or B cell related disorder, e.g., a nude mouse or a SCID mouse, including administering to the animal a nucleic acid encoding an Ikaros peptide and expressing the nucleic acid.
In another aspect, the invention features a method of evaluating the effect of a treatment, e.g., a treatment designed to promote or inhibit hematopoiesis, including carrying out the treatment and evaluating the effect of the treatment on the expression of the Ikaros gene.
In preferred embodiments the treatment is administered: to an animal, e.g., a human, a mouse, a transgenic animal, or an animal model for an immune system disorder, e.g., a T or B cell related disorder, e.g., a nude mouse or a SCID mouse, or a cell, e.g., a cultured stem cell.
In another aspect, the invention features a method for determining if a subject, e.g., a human, is at risk for a disorder related to mis-expression of the Ikaros gene, e.g., a leukemic disorder or other disorder of the immune system, e.g., an immunodeficiency, or a T or B cell related disorder, e.g., a disorder characterized by a shortage of T or B cells, including examining the subject for the expression of the Ikaros gene, non-wild type expression or mis-expression being indicative of risk.
In another aspect, the invention features a method for determining if a subject, e.g., a human, is at risk for a disorder related to mis-expression of the Ikaros gene, e.g., a leukemic disorder or other disorder of the immune system, e.g., an immunodeficiency, or a T or B cell related disorder, e.g., a disorder characterized by a shortage of T or B cells, including providing a nucleic acid sample from the subject and determining if the structure of an Ikaros gene allele of the subject differs from wild type.
In preferred embodiments: the determination includes determining if an Ikaros gene allele of the subject has a gross chromosomal rearrangement; the determination includes sequencing the subject""s Ikaros gene.
In another aspect, the invention features, a method of evaluating an animal or cell model for an immune disorder, e.g., a T cell related disorder, e.g., a disorder characterized by a shortage of T or B cells, including determining if the Ikaros gene in the animal or cell model is expressed at a predetermined level or if the Ikaros gene is mis-expressed. In preferred embodiments: the predetermined level is lower than the level in a wild type or normal animal; the predetermined level is higher than the level in a wild type or normal animal; or the pattern of isoform expression is altered from wild type.
In another aspect, the invention features a transgenic rodent, e.g., a mouse, having a transgene which includes an Ikaros gene or Ikaros protein encoding DNA. In preferred embodiments: the Ikaros gene or DNA includes a deletion, e.g. a deletion of all or part of one or more Ikaros exons, e.g., a deletion of all or part of exon E7 or a deletion of all or part of exons E3 or E4, or is otherwise mis-expressed; the Ikaros gene encodes an Ikaros protein which is a competitive antagonist of a naturally occurring Ikaros protein.
In another aspect, the invention features a method of expressing a heterologous gene, e.g., in a cell e.g., a stem cell, including placing the gene under the control of an Ikaros-responsive control element, and contacting the Ikaros-responsive control element with an Ikaros protein.
In preferred embodiments: the Ikaros-responsive control element includes an enhancer, e.g., an xcex4A element, an NFKB element, or one of the Ikaros binding sequences, e.g., one of the consensus sequences, disclosed herein; the Ikaros-responsive control element includes the regulatory region of the CD3xcex4 gene; the heterologous gene and the Ikaros-responsive control element are carried on a vector; the method further includes the step of transforming a cell with a vector which includes a heterologous gene under the control of an Ikaros-responsive control agent; the heterologous gene is expressed in a cell which normally includes or expresses an Ikaros protein.
In another aspect, the invention features a method of expressing a gene under the control of an Ikaros-responsive control element in a cell including administering an Ikaros protein to the cell.
In preferred embodiments: the method further includes transforming the cell with DNA which encodes an Ikaros protein to supply an Ikaros protein, the gene is a heterologous gene.
In another aspect, the invention features a method for treating, an animal, e.g., a human, a mouse, a transgenic animal, or an animal model for a disorder of the nervous system, e.g., a disorder of the corpus striatum, e.g., Alzheimer""s disease, immune system disorder, including administering a therapeutically effective amount of an Ikaros protein to the animal.
In another aspect, the invention features a method for treating an animal, e.g., a human, a mouse, a transgenic animal, or an animal model for a disorder of the nervous system, e.g., a disorder of the corpus striatum, e.g., Alzheimer""s disease, including administering to the animal cells selected, e.g., selected in vitro, for the expression of a product of the Ikaros gene, e.g., hematopoietic stem cells, e.g., cells transformed with Ikaros-protein-encoding DNA, e.g., hematopoietic stem cells transformed with Ikaros-protein-encoding DNA.
In preferred embodiments: the cells are taken from the animal to which they are administered; the cells are taken from an animal which is MHC matched with the animal to which they are administered; the cells are taken from an animal which is syngeneic with the animal to which they are administered: the cells are taken from an animal which is of the same species as is the animal to which they are administered.
In another aspect, the invention features a method for treating an animal, e.g., a human, a mouse, a transgenic animal, or an animal model for a disorder of the nervous system. e.g., a disorder of the corpus striatum, e.g., Alzheimer""s disease, including administering to the animal a nucleic acid encoding an Ikaros peptide and expressing, the nucleic acid.
In another aspect, the invention features a method of evaluating the effect of a treatment for a disorder of the nervous system, e.g., a disorder of the corpus striatum, e.g., Alzheimer""s disease, including administering the treatment and evaluating the effect of the treatment on the expression of the Ikaros gene.
In preferred embodiments the treatment is administered: to an animal, e.g., a human, a mouse, a transgenic animal, or an animal model for a disorder of the nervous system, e.g., a disorder of the corpus striatum, e.g., Alzheimer""s disease, or a cell, e.g., a cultured stem cell.
In another aspect, the invention features a method for determining if a subject, e.g., a human, is at risk for a disorder related to mis-expression of the Ikaros gene, e.g., a disorder of the nervous system, e.g., a disorder of the corpus striatum, e.g., Alzheimer""s disease, including examining the subject for the expression of the Ikaros gene, non-wild type expression or mis-expression being indicative of risk.
In another aspect, the invention features a method for determining if a subject, e.g., a human, is at risk for a disorder related to mis-expression of the Ikaros gene, e.g., a disorder of the nervous system, e.g., a disorder of the corpus striatum, e.g., Alzheimer""s disease, including providing a nucleic acid sample from the subject and determining if the structure of an Ikaros gene allele of the subject differs from wild type.
In preferred embodiments: the determination includes determining if an Ikaros gene allele of the subject has a gross chromosomal rearrangement; the determination includes sequencing the subject""s Ikaros gene.
In another aspect, the invention features, a method of evaluating an animal or cell model for a disorder of the nervous system, e.g., a disorder of the corpus striatum, e.g., Alzheimer""s disease, including determining if the Ikaros gene in the animal or cell model is expressed at a predetermined level or if the Ikaros gene is mis-expressed.
In preferred embodiments: the predetermined level is lower than the level in a wild type or normal animal; the predetermined level is higher than the level in a wild type or normal animal.
In another aspect, the invention features, a method of inhibiting an interaction, e.g., binding, between a protein, e.g., a first Ikaros isoform, and a DNA sequence, e.g., a DNA sequence under the control of a xcex4A sequence, an NKFB sequence, a sequence which corresponds to an Ikaros binding oligonucleotide described herein, or a site present in the control region of a lymphocyte restricted gene, e.g., TCR-xcex1, -xcex2, or -xcex4, CD3-xcex4, -xcex5, -xcex3 genes, the SL3 gene, or the HIV LTR gene. The methods includes contacting the DNA sequence with an effective amount of a second Ikaros isoform, or with a DNA binding fragment encoding an Ikaros isoform, e.g., of the second Ikaros isoform, e.g. of an Ikaros antagonist isoform.
In preferred embodiments: the fragment is deleted for all or part of an Ikaros exon, e.g., for all or part of E1/2, E3, E4, E5, E6, or E7; the antagonist contains one or more point mutations relative to a naturally occurring Ikaros isoform; the antagonist comprises at least two different exons which are sequentially oriented, in the antagonist, in a permuted (e.g. non wild-type) fashion relative to naturally occurring Ikaros isoforms (e.g. any of SEQ. NOS: 2-8); the antagonist comprises at least two of the same Ikaros exon.
In another aspect, the invention features, a method of inhibiting an interaction, e.g., binding, between a protein, e.g., a first Ikaros isoform, and a DNA sequence, e.g., a xcex4A sequence, an NKFB sequence, a sequence which corresponds to an Ikaros binding oligonucleotide described herein, or a site present in the control region of a lymphocyte restricted gene, e.g., TCR-xcex1, -xcex2, or -xcex4, CD3-xcex4, -xcex5, -xcex3 genes, the SL3 gene, or the HIV LTR gene. The methods includes contacting the protein with an effective amount of an Ikaros binding oligonucleotide. In preferred embodiments the oligonucleotide includes a sequence chosen from, IK-BS1, IK-BS2, IK-BS3, IK-BS4, IK-BS5, IK-BS6, IK-BS7, IK-BS8, or IK-BS9.
In preferred embodiments: the oligonucleotide preferentially binds to a first Ikaros isoform; the oligonucleotide preferentially binds to a second Ikaros isoform.
In another aspect the invention includes an Ikaros binding oligonucleotide, e.g., IK-BS 1, IK-BS2, IK-BS3, IK-BS4, IK-BS5, IK-BS6, IK-BS7, IK-BS8, or IK-BS9. In preferred embodiments the oligonucleotide contains at least two, three, four, or five copies of one of the Ikaros binding oligonucleotide sequences disclosed herein.
In another aspect, the invention features a method of attenuating the binding of a first Ikaros isoform to target DNA. The method includes contacting the target DNA with an effective amount of a second Ikaros isoform, or with a DNA binding fragment of said second isoform. The second Ikaros isoform can be, for example, an antagonists isoform of Ikaros, e.g. an Ikaros isoform generated point mutation, e.g. an Ikaros isoform generated by in vitro exon shuffling.
In another aspect, the invention features a method of modulating the rate of division or amplification of a cell, or entry of the cell into the cell cycle. The method includes administering to the cell, an effective amount of an Ikaros polypeptide, or a nucleic acid encoding an Ikaros polypeptide. The method can be practiced ex vivo, in vivo, or in vitro.
In preferred embodiments, the cell is a hematopoietic cell, e.g., a stem cell, e.g., a totipotent or a pluripotent stem cell, or a descendent of a stem cell, e.g., a lymphocyte. In preferred embodiments the cell is a human, a pig, a rabbit, or a rodent, e.g., a mouse or rat, cell.
In preferred embodiments the division or amplification, or entry into the cell cycle, is promoted. Generally, Ikaros mutations which inhibit or antagonize normal non-proliferative Ikaros function (e.g., the function of the Ik-1 isoform) increase cell division. Such mutants include: mutations which inhibit DNA binding, e.g., point mutations in, or deletions for all or part of, one or more of F1 , F2, F3, or F4; mutations. e.g., point mutations in, or deletions for all or part of, one or more of exons 1/2, 3, 4, 5, or 6; mutations which results in the preferential expression of proliferation-promoting Ikaros dimer subunits as opposed to non-proliferation-promoting Ikaros dimer subunits; or mutants having defective DNA binding but functional dimerization domains. Less preferred are mutations which inactivate one or both of transcriptional activation or dimerization, which decrease the half life of the protein, or which inactivate one or both of the C terminal Zinc finger domains, e.g., F5 or F6; or a mutation is a C terminal deletion. Fragments or other mutants of Ikaros (or Aiolos) which inhibit dimerization of Ikaros proteins, e.g., fragments which include the C terminal dimerization region, e.g., fragments which include Zinc fingers F5 and F6, can also be used to promote cell division. Subunits of proliferation-promoting Ikaros dimers can also increase division, amplification, or entry into the cell cycle.
Methods for increasing cell division can be combined with procedures where it is desirable to increase cell division, e.g., the treatment, e.g., by chemotherapy or radiotherapy, of tumors or other cell-proliferative disorders.
In preferred embodiments the division, amplification, or entry into the cell cycle is decreased. Subunits of non-proliferation-promoting Ikaros dimers, e.g., Ik-1, can decrease division, amplification, or entry into the cell cycle.
The cell can be implanted into a mammal, e.g., into the mammal from which it was derived, or into a mammal of the same or a different species. The cell can be cultured prior to introduction into the mammal. E.g., the cell can be altered to modulate the rate of division or amplification of the cell, or entry of the cell into the cell cycle, and then implanted into the mammal. The mammal can be, e.g., a human, a non-human primate, a pig, a rat, a rabbit, or a rodent, e.g., a rat or mouse.
The invention also includes a cell, a purified preparation of cells, or an ex vivo preparation of cells, in which the rate of division or amplification of the cell, or entry of the cell into the cell cycle, has been modulated, e.g., changes as compared to a wild type or non-modulated cell of equivalent type.
In another aspect, the invention features a method of modulating the state of differentiation of a cell. The method includes administering to the cell, an Ikaros polypeptide, or a nucleic acid encoding an Ikaros polypeptide, in an amount sufficient to modulate, e.g., to promote the maintenance of the state of differentiation of the cell, or to promote differentiation. The method can be practiced ex vivo, in vivo, or in vitro.
In preferred embodiments, the cell is a hematopoietic cell, e.g., a stem cell, e.g., a totipotent or a pluripotent stem cell, or a descendent of a stem cell, e.g., a lymphocyte. In preferred embodiments the cell is a human, a pig, a rabbit, or a rodent, e.g., a mouse or rat,
In preferred embodiments, the state of differentiation of the cell is maintained, e.g., differentiation is inhibited and a more primitive and more multipotent state is promoted. This can be achieved by providing Ikaros polypeptides having wild type non-proliferative function, e.g., Ikaros polypeptides having the function of the IK-1 isoform. Subunits of non-proliferation-promoting Ikaros dimers can promote maintenance of the differentiated state of the cell. In a particularly preferred embodiment wild type Ikaros function is provided to human hematopoietic cells, preferably stem cells, to maintain their differentiated state or to otherwise enhance culturing of the cells.
In preferred embodiments, differentiation of the cell, which is usually accompanied by entry into the cell cycle, is promoted. Generally, Ikaros mutations which inhibit or antagonize normal non-proliferative Ikaros function (e.g., the function of the Ik-1 isoform) promote differentiation. Such mutants include: mutations which inhibit DNA binding, e.g., point mutations in, or deletions for all or part of, one or more of F1, F2, F3, or F4; mutations e.g., point mutations in, or deletions for all or part of, one or more of exons 1/2, 3, 4, 5, or 6; mutations which results in the preferential expression of proliferation-promoting Ikaros dimer subunits as opposed to non-proliferation-promoting Ikaros dimer subunits; or mutants having defective DNA binding but functional dimerization domains. Less preferred for promoting differentiation are mutations which inactivate one or both of transcriptional activation or dimerization, which decrease the half life of the protein, or which inactivate one or both of the C terminal Zinc finger domains, e.g., F5 or F6; or a mutation is a C terminal deletion. Fragments or other mutants of Ikaros (or Aiolos) which inhibit dimerization of non-proliferative Ikaros proteins, e.g., fragments which include the C terminal dimerization region, e.g., fragments which include Zinc fingers F5 and F6, can also be used to promote differentiation. Subunits of proliferation-promoting Ikaros dimers can be used to promote differentiation.
The cell can be implanted into a mammal, e.g., into the mammal from which it was derived, or into a mammal of the same or a different species. The cell can be cultured prior to introduction into the mammal. E.g., the cell can be altered to modulate the state of differentiation of the cell, and then implanted into the mammal. The mammal can be, e.g., a human, a non-human primate, a pig, a rat, a rabbit, or a rodent, e.g., a rat or mouse.
The invention also includes a cell, a purified preparation of cells, or an ex vivo preparation of cells, in which the state of differentiation of the cell, has been modulated, e.g., changes as compared to a wild type or non-modulated cell of equivalent type.
In another aspect, the invention features a cell e.g., a hematopoietic cell, e.g., a stem cell, e.g., a totipotent or a pluripotent stem cell, or a descendent of a stem cell, e.g., a lymphocyte, which overexpresses an Ikaros dimer or isoform, preferably an NPID or an NPID isoform, e.g., the Ik-1 isoform. Also included in this aspect of the invention are purified or ex vivo preparations of the cells.
In preferred embodiments the cell is a human, a pig, a rabbit, or a rodent, e.g., a mouse or rat, cell.
In preferred embodiments, the NPID is e.g., a Ik-1/Ik-1, Ik-1/Ik-2, Ik-1/Ik-3, Ik-2/Ik-2, Ik-2/Ik-3 or Ik-3/Ik-3 dimer.
The cell can be implanted into a mammal, e.g., into the mammal from which it was derived, or into a mammal of the same or a different species. The cell can be cultured prior to introduction into the mammal. E.g., the cell can be altered to overexpress an Ikaros dimer or isoform and then implanted into the mammal. The mammal can be, e.g., a human, a non-human primate, a pig, a rat, a rabbit, or a rodent, e.g., a rat or mouse.
In another aspect, the invention features a cell, e.g., a hematopoietic cell, e.g., a stem cell, e.g., a totipotent or a pluripotent stem cell, or a descendent of a stem cell, e.g., a lymphocyte, in which the ratio of an NPID or NPID isoform to a PPID or PPID isoform (or generally of NPID""s to PPID""s) has been altered, e.g., increased or decreased, with respect to a wild-type or unaltered cell. Also included in this aspect of the invention are purified or ex vivo preparations of the cells.
In preferred embodiments the cell is a human, a pig, a rabbit, or a rodent, e.g., a mouse or rat, cell.
In preferred embodiments, the NPID is e.g., a Ik-1/Ik-1, Ik-1/Ik-2, Ik-1/Ik-3, Ik-2/Ik-2, Ik-3 or Ik-3/Ik-3 dimer.
In preferred embodiments, the PPID includes at least one Ikaros isoform which lacks one or more functional F1, F2, F3, or F4 zinc finger regions, e.g., Ik-4, Ik-5, Ik-6, Ik-7 or Ik-8.
In preferred embodiments, the ratio of an NPID or NPID isoform to a PPID or PPID isoform is increased, e.g., by increasing the amount of NPID or NPID isoform (relative to a PPID or PPID isoform) or by decreasing the amount of a PPID or PPID isoform (relative to an NPID or NPID isoform).
The cell can be implanted into a mammal, e.g., into the mammal from which it was derived, or into a mammal of the same or a different species. The cell can be cultured prior to introduction into the mammal. E.g., the cell can be altered with respect to the NPID/PPID ratio and then implanted into the mammal. The mammal can be, e.g., a human, a non-human primate, a pig, a rat, a rabbit, or a rodent, e.g., a rat or mouse.
A purified preparation of cells, is a preparation of cells which includes at least 10, 30, 50, 75, 90, 95, or 99%, by number or weight, the subject cells. An ex vivo preparation is a preparation of cells from a mammal or from cell culture which is suitable fro re-introduction into the same or a different mammal.
Heterologous gene, as used herein, is a gene which is not normally under the control of an Ikaros responsive control element.
An Ikaros-responsive control element, as used herein is a region of DNA which, when present upstream or downstream from a gene, results in regulation, e.g., increased transcription of the gene in the presence of an Ikaros protein.
Purified DNA is DNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (i.e., one at the 5xe2x80x2 end and one at the 3xe2x80x2 end) in the naturally occurring genome of the organism from which the DNA of the invention is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector: into an autonomously replicating plasmid or virus: or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
Homologous refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences. For example, 6 of 10, of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology.
A transgene is defined as a piece of DNA which is inserted by artifice into a cell and becomes a part of the genome of the animal which develops in whole or part from that cell. Such a transgene may be partly or entirely heterologous to the transgenic animal.
A transgenic animal, e.g., a transgenic mouse, is an animal having cells that contain a transgene, which transgene was introduced into the animal, or an ancestor of the animal, at a prenatal, e.g., an embryonic stage.
An enhancer region is defined as a cis-acting DNA sequence capable of increasing transcription from a promoter that is located either upstream or downstream of the enhancer region. Such DNA sequences are well known to those skilled in the art of eukaryotic gene expression.
A substantially pure preparation of a peptide is a preparation which is substantially free of one or more of the peptides with which it naturally occurs in a cell. A substantially pure preparation of a non-naturally occurring peptide is one which is at least 10% by weight of the peptide of interest. In a preferred embodiment, a substantially pure preparation further lacks any nucleic acids, such as oligonucleotides, which bind to the subject Ikaros protein.
Mis-expression, as used herein, refers to a non-wild type pattern of gene expression. It includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of the tissue specificity of expression, e.g., increased or decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the size, amino acid sequence, post-translational modification, or a biological activity of an Ikaros gene product; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellullar stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus; or a pattern of isoform expression which differs from wild type.
The terms peptide, protein, and polypeptide are used interchangeably herein.
A peptide has Ikaros activity if it has one or more of the following properties: the ability to stimulate transcription of a DNA sequence under the control any of a xcex4A element, an NFKB element, or one of the Ikaros binding oligonucleotide consensus sequences disclosed herein; the ability to bind to any of a xcex4A element, an NFKB element, or one of the Ikaros binding oligonucleotide consensus sequences disclosed herein; or the ability to competitively inhibit the binding of a naturally occurring Ikaros isoform to any of a xcex4A element, an NFKB element, or one of the Ikaros binding oligonucleotide consensus sequences disclosed herein. An Ikaros peptide is a peptide with Ikaros activity.
As used herein, the term xe2x80x9cexonxe2x80x9d, refers to those gene (e.g. DNA) sequence which are transcribed and processed to form mature messenger RNA (mRNA) encoding an Ikaros protein, or portion thereof, e.g. Ikaros coding sequences, and which, at the chromosomal level, are interrupted by intron sequences. Exemplary exons of the subject Ikaros proteins and genes include: with reference to SEQ ID NO:5 (mIk-1), the nucleotide sequence encoding exon 1/2 (E1/2) corresponding to Met-1 through Met-53 (encoded by nucleotides 223 to 381 of SEQ ID NO:5); the nucleotide sequence encoding exon 3 (E3) corresponding to Ala-54 through Thr 140 (encoded by nucleotides 382 to 642 of SEQ ID NO:5); the nucleotide sequence encoding exon 4 (E4) corresponding to Gly-141 through Ser-196 (encoded by nucleotides 643 to 810 of SEQ ID NO:5); the nucleotide sequence encoding exon 5 (E5) corresponding to Val-197 through Pro-237 (encoded by nucleotides 811 to 933 of SEQ ID NO:5); the nucleotide sequence encoding exon 6 (6) corresponding to Val-238 through Leu-282 (encoded by nucleotides 934 to 1068 of SEQ ID NO:5); the nucleotide sequence encoding exon 7 (E7) corresponding to Gly-283 through Ser-518 (encoded by nucleotides 1069 to 1776 of SEQ ID NO:5), with reference to SEQ ID NO:3 (1-Ik-1), the nucleotide sequence encoding exon 3 (E3) corresponding to Asn-1 through Thr-85 (encoded by nucleotides 1 to 255 of SEQ ID NO:3); the nucleotide sequence encoding exon 4 (E4) corresponding to Gly-86 through Ser-141 (encoded by nucleotides 256 to 423 of SEQ ID NO:3); the nucleotide sequence encoding exon 5 (E5) corresponding to Val-142 through Pro-183 (encoded by nucleotides 424 to 549 of SEQ ID NO:3); the nucleotide sequence encoding exon 6 (6) corresponding to Val-184 through Leu-228 (encoded by nucleotides 550 to 684 of SEQ ID NO:3); the nucleotide sequence encoding exon 7 (E7) corresponding to Gly-229 through Ser-461 (encoded by nucleotides 685 to 1383 of SEQ ID NO:3). The term xe2x80x9cintronxe2x80x9d refers to a DNA sequence present in a given Ikaros gene which is not translated into protein and is generally found between exons. The term xe2x80x9cgenexe2x80x9d refers to a region of chromosomal DNA which contains DNA sequences encoding an Ikaros protein, including both exon and intron sequences. A xe2x80x9crecombinant genexe2x80x9d refers to nucleic acid encoding an Ikaros protein and comprising Ikaros exon sequence, though it may optionally include intron sequences which are either derived from a chromosomal Ikaros gene or from an unrelated chromosomal gene. An exemplary recombinant gene is a nucleic acids having a sequence represented by any of SEQ ID NOS:2-8 or 153 or SEQ ID NO:165.
The term xe2x80x9cIkaros responsive elementxe2x80x9d or xe2x80x9cIK-RExe2x80x9d, refers to nucleic acid sequences which, when placed in proximity of a gene, act as transcriptional regulatory elements which control the level of transcription of the gene in an Ikaros protein-dependent manner. Exemplary IK-RE, as described below, include IK-BS1, IK-BS2, IK-BS4, IK-BS5, IK-BS6, IK-BS7, IK-BS8, or IK-BS9.
A xe2x80x9cnon-proliferative Ikaros dimerxe2x80x9d (NPID), e.g., Ik-1/Ik-1, Ik-1/Ik-2, Ik-1/Ik-3, Ik-2/Ik-2, Ik-2/Ik-3 or Ik-3/Ik-3, inhibits proliferation of the cell. A xe2x80x9cproliferative Ikaros dimerxe2x80x9d (PPID), promotes proliferation and includes at least one Ikaros isoform which lacks one or more functional F1, F2, F3, or F4 zinc finger regions, e.g., Ik-4, Ik-5, Ik-6, Ik-7 or 8. Proliferation can mean proliferation as compared to an otherwise similar cell or as compared to a wild-type cell. The concentration or activity of an NPID or PPID can be manipulated by any means known to the art. For example, the concentration or activity of an NPID can be reduced by reducing the availability of one or more monomeric species which can form an NPID, e.g., by reducing the availability of one or more of Ik-1, Ik-2, or Ik-3. Such reduction can be effected by mutations which decrease production of Ik-1, Ik-2, or Ik-3, by the expression of antisense molecules which inhibit Ik-1, Ik-2, or Ik-3 expression or by compounds which inhibit dimerization of the subunits of NPID""s. The concentration or activity of NPID""s can be reduced by providing Ikaros species which lack one or more functional F1, F2, F3, or F4 zinc finger regions, e.g., by producing Ik-4, Ik-5, Ik-6, or Ik-7 isoforms. Such species can form proliferation-promoting Ikaros dimers (PPID). Thus, manipulations which reduce the concentration or activity of NPID, e.g., by sequestering available Ik-1, Ik-2, or Ik-3 in PPID""s, can be used to provide an Ikaros-deregulated lymphocyte. The concentration of an NPID can be increased by providing isoforms which form NPID""s. The ratio of NPID""s/PPID""s can be increased by decreasing the concentration of PPID""s.
The invention is useful for identifying T cells; identifying cells which can develop into T cells; and generally, in the investigation of hemopoiesis, e.g., in the differentiation of progenitor stem cells into T cells. The role of the Ikaros gene and its products can be studied, e.g., in cells, e.g., cultured cells, transformed with the Ikaros gene or fragments thereof, or in transgenic animals. The invention is also useful for: promoting the expression of markers of cell lineage, e.g., CD3xcex4 genes; enhancing the ability of a cell, e.g., a stem cell, to develop into a T cell; screening individuals at risk for genetic T cell disorders, e.g., leukemia; and treating immune disorders (e.g., immunodeficiencies, e.g., AIDS, or chemical, drug, or radiation induced immunodeficiencies, or cancers, e.g., leukemia) characterized by a shortage of T cells; for investigating the structure and expression of the Ikaros gene or iso forms of the gene product; for investigating species or tissue differences in the expression of the Ikaros gene or its isoforms; for investigating the structure and function of DNA binding proteins; for studying the structure and function of zinc finger containing proteins; for the construction of transgenic animals; for inhibiting the binding of Ikaros to a target molecule; for studying the relative affinities of Ikaros isoforms for target DNA; and for searching for or manipulating the expression of genes under the control of Ikaros isoforms.
Other features and advantages of the invention will be apparent from the following description and from the claims.